The present invention relates to a method f or quantitative determination of bilirubin and a reagent composition for quantitative determination of bilirubin, which are useful in clinical examinations.
Bilirubin is a physiological metabolite of heme and is a dye which is present most largely in bile. In serum, it exists in the direct form and in the indirect form, and it is extremely important to differentially determine the amount of direct bilirubin and indirect bilirubin in clinical examinations. The conventional method for differential determination of direct bilirubin and indirect bilirubin which has been carried out for a long time in clinical examinations, involves determination of total bilirubin and determination of direct bilirubin, and the indirect bilirubin is obtained by subtracting the direct bilirubin from the total bilirubin.
In quantitative determination of bilirubin by a chemical means, known is a method of colorimetrically measuring the amount of azobilirubin to be formed through the reaction of bilirubin with a diazo reagent. However, the method is problematic in that the operation is complicated and in that accurate data have not been obtained since the diazo reagent used reacts with some organic compounds other than bilirubin in sample.
On the other hand, in quantitative determination of bilirubin by an enzymatic means, known are methods using bilirubin oxidase derived from fungi belonging to the genus of Basidiomyctes (Japanese Published Unexamined Patent Application No. 27656/81) and microorganisms belonging to the genus of Myrothecium (Japanese Published Unexamined Patent Application No. 159487/82) which catalyze the oxidation of bilirubin. In the methods, standard solutions having varying concentrations of bilirubin to be reacted with the bilirubin oxidase give a calibration curve showing the relationship between the bilirubin concentration and the change in absorbance of the reaction mixture, and a sample having an unknown bilirubin concentration is reacted with the oxidase to give the concentration calculated by comparison of the measured change in absorbance with the calibration curve.
There has also been reported a method of quantitative determination of total bilirubin and direct bilirubin that uses an enzyme selected from the group consisting of bilirubin oxidase, ascorbate oxidase, laccase and tyrosinase, in the presence of a reaction promoter selected from the group consisting of surfactants, aromatic carboxylates, sulfa drugs and proteases (Japanese Published Unexamined Patent Application No. 17999/84).
The quantitative determination of bilirubin using enzyme is defective in the following points. Since bilirubin oxidase is unstable in an aqueous medium, a reagent containing bilirubin oxidase is often prepared in the form of a freeze-dried powder. The reagent must be dissolved in an aqueous medium just before use and the life of the prepared reagent is short. The other oxidases except bilirubin oxidase, such as ascorbate oxidase, etc., do not effectively oxidize bilirubin. Even if some known reaction promoters are added to these reagents, the oxidation of bilirubin is not promoted very much.